1. Field of the Invention
This invention relates to apparatus for screening compound libraries, such as compound libraries generated using combinatorial chemistry techniques. The apparatus of this invention employ frontal chromatography in combination with mass spectrometry to screen a library of compounds to identify and rank those members of the library that bind to a target receptor. The apparatus of this invention also permit a compound library to be rapidly screened to determine if one or more members of the library have an affinity for a target receptor as measured by a pre-selected indicator compound.
The following publications, patents and patent applications are cited in this application as superscript numbers:
1 K. S. Lam, Anti-Cancer Drug Des. 1997, 12, 145-167.
2 P. M. Sweetnam et al., In Burger""s Medicinal Chemistry and Drug Discovery; M. E. Wolff, Ed.; John Wiley and Sons: New York, 1995; pp 697-731.
3 R. H. Griffey et al., In Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, Calif., Jun. 1-5, 1997; p. 400.
4 L. Fang et al., In Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, Calif., Jun. 1-5, 1997; p. 401.
5 Y. -H. Chu et al., J. Am. Chem. Soc. 1996, 118, 7827-7835.
6 Y. -Z. Zhao et al., J. Med. Chem. 1997, 40, 4006-4012.
7 Y. F. Hsieh et al., J. Mol. Div. 1996, 2, 189-196.
8 R. W. Nelson et al., Anal. Chem. 1995, 67, 1153-1158.
9 D. C. Schriemer and L. Li, Anal. Chem. 1996, 68, 3382-3387.
10 PCT/US97/07964 (International Publication No. WO 97/43641), published Nov. 20, 1997, entitled xe2x80x9cMolecular Diversity Screening Device and Method.xe2x80x9d
11 R. Wieboldt et al., Anal. Chem. 1997, 69, 1683-1691.
12 R. B. van Breemen et al., Anal. Chem. 1997, 69, 2159-2164.
13 M. L. Nedved et al., Anal. Chem. 1996, 68, 4228-4236.
14 PCT/US95/03355 (International Publication No. WO 95/25737), published Sep. 28, 1995, entitled xe2x80x9cMethod for Identifying Members of Combinatorial Libraries.xe2x80x9d
15 PCT/EP97/02215 (International Publication No. WO 97/43301), published Nov. 20, 1997, entitled xe2x80x9cIdentification of Members of Combinatorial Libraries By Mass Spectrometry.xe2x80x9d
All of the above publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
2. State of the Art
In recent years, a large number of combinatorial chemistry techniques have been developed which permit vast libraries of diverse chemical compounds to be rapidly synthesized.1 In combinatorial chemistry, a series of chemical reactions is typically conducted employing a plurality of reagents at each step to generate a library of compounds. Such techniques have the potential to greatly accelerate the discovery of new compounds having biologically useful properties by providing large collections of diverse chemical compounds for biological screening.
This ability to rapidly generate large collections of compounds using combinatorial chemistry techniques has created a need for new methods of screening compound libraries. The traditional approach of screening each compound individually in an assay to identify those compounds having the desired biological activity is no longer practical due to time and resource constraints. Thus, a need exists for new methods and apparatus which permit the rapid screening of compound libraries.
In this regard, various methods for screening compound libraries have been reported. Typically, these screening methods involve the use of target receptors which have been labeled with fluorescent or other reporter groups.2 In these methods, the compound library, typically bound to a resin bead, is exposed to the labeled target receptor and those members binding to the labeled target receptor are identified and physically separated. The particular ligand binding to the target receptor is then identified. In many of these techniques, elaborate procedures are required to keep track of individual members of the library. For example, coded tags are often added during the synthesis of the combinatorial library to allow the structure of the individual members to be subsequently determined. Alternatively, combinatorial libraries can be prepared in an array and the individual members of the library subsequently identified by their location in the array. While such methods can be effective, the need to keep track of individual members of the library during their synthesis and screening is quite cumbersome and often limits the type of synthetic procedures that can be employed. Additionally, many of these techniques require that the synthetic procedures be conducted on a solid phase, thus further limiting the synthetic procedures and reagents that can be used.
As an alternative, mass spectrometry is emerging as an important tool for the interrogation of combinatorial libraries. To date, mass spectrometry has been used to assess library quality3, 4 and, when coupled with molecular recognition technologies, has allowed for some success in the isolation and characterization of active library compounds.5-15 Typically, when screening compound libraries for biologically active members, mass spectrometry is used in combination with a xe2x80x9ccapture and releasexe2x80x9d methodology. In this methodology, compound mixtures are presented to the target receptor, which is often immobilized on a solid support, and the resulting ligand-receptor complexes are separated from the unbound members of the library. After separation, the ligand-receptor complexes are typically denatured, for example, with a solvent and the solvent mixture containing the previously bound ligands is presented to the mass spectrometer to permit identification of the high affinity ligands.
For example, ultrafiltration has been used in combination with electrospray mass spectrometry to screen combinatorial libraries.10-12 In this method, ligands present in a compound library are allowed to bind to a receptor and the resulting ligand-receptor complexes are purified by ultrafiltration. The ligand-receptor complexes are then dissociated using a solvent, such as methanol, and the previously bound ligands are detected by an electrospray mass spectrometer.
Affinity capillary electrophoresis (ACE) has also been coupled with mass spectrometry to screen combinatorial libraries.5 In this procedure, ACE is used to separate ligand-receptor complexes from unbound ligands and mass spectrometry is used to identify the high affinity ligands.
Similarly, compound libraries have been screened using affinity chromatography in combination with mass spectrometry. For example, WO 97/43301 describes a method for characterizing the members of a combinatorial library, which method utilizes affinity selection in combination with mass spectrometry. Specifically, the members of the library are brought into contact with a domain of interest to allow for binding, i.e., the formation of a complex. After binding, the complex is separated from the unbound members of the library, typically by washing the unbound members from the column containing the complexes. The complexes are then treated to elute the bound library components and the eluted components are analyzed by mass spectrometry. The elution methods described include the use of displacers, chaotrope agents, pH elution, salt gradients, temperature gradients, organic solvents, selective denaturants and detergents. Using such methods, the weakly bound members of the library are purportedly eluted first and analyzed by mass spectrometry, followed by the elution of the more strongly bound members.
There are several disadvantages associated with the xe2x80x9ccapture and releasexe2x80x9d methods for screening compound libraries that have been previously reported. First, the procedure used to xe2x80x9creleasexe2x80x9d the bound ligands from the ligand-receptor complexes may alter the binding profile for the various bound ligands, resulting in a false indication of binding strength. For example, using a pH gradient to release the bound members of the library may change the electronic character of the binding site on the receptor causing ligands which are strongly bound under physiological conditions to be prematurely released. Thus, the characterization of binding strength for various ligands based on their relative time of release may be misleading if the release conditions are different from the binding conditions. Accordingly, these methods may not accurately identify the most active members of a compound library. Additionally, certain conditions used for compound release, such as pH gradients, may irreversibly denature the receptor thus preventing its subsequent use for screening compound libraries.
Additionally, when xe2x80x9ccapture and releasexe2x80x9d methods are employed, each bound ligand is typically released over a relatively short period of time resulting, for example, in an elution peak or xe2x80x9cspikexe2x80x9d for each ligand. Accordingly, the effluent produced using such methods is typically monitored continually, for example, by mass spectrometry so that any particular elution peak is not missed. Thus, the number of analyses that can be conducted using any particular mass spectrometer is limited. Accordingly, it would be desirable to develop methods and apparatus for screening compound libraries that do not rely upon xe2x80x9ccapture and releasexe2x80x9d methodologies.
This invention is directed to apparatus for screening compound libraries. The compound libraries may be generated or obtained by any means including, by way of example, combinatorial chemistry techniques or from fermentation broths, plant extracts, cellular extracts and the like. The apparatus of this invention employ frontal chromatography (FC) in combination with mass spectrometry (MS) to screen the library of compounds to identify and rank those members of the library that bind to a target receptor.
In frontal chromatography, a target receptor is typically immobilized on a suitable solid support material and packed in a column. A mixture containing putative ligands is then continuously infused through the column. Ligands having an affinity for the target receptor bind to the column, but eventually the capacity of the column for each ligand is exceeded and the ligands elute or xe2x80x9cbreak throughxe2x80x9d at their infusion concentration. Once a ligand begins eluting from the column, it is continually present in the effluent. Compounds having little or no affinity for the target receptor break through earlier in the effluent compared to ligands having a higher affinity for the receptor.
In the present invention, mass spectrometry (MS) is employed to continuously or intermittently monitor the FC effluent. Using MS, the identity and break through time for each ligand on the column can be determined. Thus, FC-MS allows the relative affinity of each member of the library for the target receptor to be determined relative to other members of the library under ligand-receptor binding conditions. Using the present apparatus, an accurate ranking of the relative affinity of each member of the compound library for the target receptor can be ascertained.
Accordingly, in one of its apparatus aspects, the present invention is directed to an apparatus for screening a compound library to determine the relative or absolute affinity of a plurality of putative ligands to a target receptor or a plurality of target receptors, which apparatus comprises:
(a) a column comprising a target receptor or a plurality of target receptors, each target receptor optionally attached to a solid phase support, and having a inflow end and an outflow end, wherein said column is capable of having a compound library comprising a plurality of putative ligands applied thereto under frontal chromatography conditions to produce an effluent from the outflow end of the column;
(b) a first reservoir connected to the inflow end of said column for applying the compound library to the column;
(c) a mass spectrometer connected to the outflow end of said column for continuously or intermittently analyzing the effluent from the column.
In a preferred embodiment, the above described apparatus further comprises:
(d) a second reservoir connected to the inflow end of the column for applying either (i) a mixture comprising the compound library, at least one void marker compound and an indicator compound or a plurality of indicators compounds, (ii) at least one void marker compound and an indicator compound or a plurality of indicator compounds, or (iii) a buffer solution to the column.
In another preferred embodiment, the above described apparatus further comprises:
(e) a third reservoir connected to the outflow end of the column for supplying a supplemental diluent to the effluent before analysis by the mass spectrometer.
Preferably, the column employed in this invention will have an internal diameter (i.d.) ranging from about 10 xcexcm to about 4.6 mm. More preferably, the internal diameter of the column will be in the range of from about 100 xcexcm to about 250 xcexcm.
Preferably, the column will range in length from about 1 cm to about 30 cm, more preferably from about 2 cm to about 20 cm.
Preferably, each target receptor is independently selected from the group consisting of proteins, including recombinant proteins, glycoproteins, glycosaminoglycans, proteoglycans, integrins, enzymes, lectins, selecting, cell-adhesion molecules, toxins, bacterial pili, transport proteins, receptors involved in signal transduction or hormone-binding, hormones, antibodies, major histocompatability complexes, immunoglobulin superfamilies, cadherins, DNA or DNA fragments, RNA and RNA fragments, whole cells, cell fragments, tissues, bacteria, fungi, viruses, parasites, preons, and synthetic analogs or derivatives thereof.
Additionally, each target receptor is preferably bound to a solid phase support. More preferably, each target receptor is covalently bound to the solid phase support or bound via biotin-avidin or biotin-streptavidin binding.
Preferably, the solid phase support used in this invention is selected from the group consisting of polymeric (resin) beads, polymeric gels, glass beads, silica chips, silica capillaries, agarose, diatomaceous earths and pulp.
The column employed in this invention preferably contains from about 1 fmol to about 10 nmol of target receptor-active sites; preferably, from about 1 pmol to about 10 nmol of target receptor active sites.
Preferably, the mass spectrometer employed in this invention is an electrospray mass spectrometer.
Additionally, since FC-MS does not require constant effluent monitoring, a plurality of FC-MS analyses can be conducted simultaneously using a single mass spectrometer to intermittently monitor each column. Furthermore, under FC conditions, since ligands are always present in the effluent once they break through the column, the intermittent monitoring of each column does not necessarily require monitoring the actual break through time for each ligand. Therefore, a plurality of FC-MS analyses can be conducted simultaneously using a single mass spectrometer to intermittently monitor each column.
Accordingly, in another of its apparatus aspects, this invention provides an apparatus for screening a plurality of compound libraries to determine the relative or absolute affinity of a plurality of putative ligands in each library to a target receptor or a plurality of target receptors, which apparatus comprises:
(a) a plurality of columns each column comprising a target receptor or a plurality of target receptors, each target receptor optionally attached to a solid phase support, and each column having a inflow end and an outflow end, wherein each of said columns is capable of independently having a compound library comprising a plurality of putative ligands applied thereto under frontal chromatography conditions to produce an effluent from the outflow end of the column;
(b) a plurality of first reservoirs each connected to the inflow end of one of the columns for applying a compound library to the columns;
(c) a mass spectrometer connected to the outflow end of each of said columns for intermittently analyzing the effluent from each of the column.
In a preferred embodiment, the above described apparatus further comprises:
(d) a plurality of second reservoirs each connected to the inflow end of one of the columns for applying either (i) a mixture comprising the compound library, at least one void marker compound and an indicator compound or a plurality of indicator compounds, (ii) at least one void marker compound and an indicator compound or a plurality of indicator compounds, or (iii) a buffer solution to the column.
In another preferred embodiment, the above described apparatus further comprises:
(e) a third reservoir connected to the outflow end of each of the columns for supplying a supplemental diluent to the effluent from each column before analysis by the mass spectrometer.
Preferably, the above described apparatus comprises from 2 to about 100 columns, more preferably from 3 to about 50 columns; and still more preferably from 5 to about 10 columns.
Preferably, each column is intermittently monitored for a period of about 0.5 seconds to about 10 seconds, preferably for about 1 second to about 5 seconds, before switching to the next column.
The apparatus of this invention can also be employed to screen a target receptor or a plurality of target receptors for affinity to an immobilized ligand or plurality of ligands. This embodiment is particularly useful for target validation studies on ligands having biological effects. Accordingly, in another of its apparatus aspects, this invention provides an apparatus for screening a target receptor or a plurality of target receptors to determine the relative affinity of the receptor or receptors to an immobilized ligand or ligands relative to an indicator compound or a plurality of indicator compounds, which apparatus comprises:
(a) a column comprising a ligand or a plurality of ligands wherein each ligand is bound to a solid phase support, said column having a inflow end and an outflow end and further wherein said column is capable of having a target receptor or a plurality of target receptors applied thereto under frontal chromatography conditions to produce an effluent from the outflow end of the column;
(b) a first reservoir connected to the inflow end of said column for applying the target receptor or receptors to the column;
(c) a second reservoir connected to the inflow end of the column for applying either (i) a mixture comprising the target receptor or receptors, at least one void marker compound and an indicator compound or a plurality of indicators compounds, (ii) at least one void marker compound and an indicator compound or a plurality of indicator compounds, or iii) a buffer solution to the column;
(d) a mass spectrometer connected to the outflow end of said column for continuously or intermittently analyzing the effluent from the column.
In a preferred embodiment, the above apparatus further comprises:
(e) a third reservoir connected to the outflow end of the column for supplying a supplemental diluent to the effluent before analysis by the mass spectrometer.
In a preferred embodiment, each ligand employed in the above apparatus is selected from the group consisting of carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, nucleosides, nucleotides, oligonucleotides, polynucleotides, lipids, retinoids, steroids, glycopeptides, glycoproteins, glycolipids, proteoglycans, and synthetic analogs or derivatives thereof.
In another preferred embodiment, each ligand is selected from the group consisting of synthetic small molecule organic compounds.
A plurality of such FC-MS analyses can also be conducted simultaneously using a single mass spectrometer to intermittently monitor each column. Accordingly, in yet another of its apparatus aspects, the present invention provides an apparatus for screening a plurality of target receptors to determine the relative affinity of the receptors to an immobilized ligand or ligands relative to an indicator compound or a plurality of indicator compounds, which apparatus comprises:
(a) a plurality of columns each column comprising a ligand or a plurality of ligands wherein each ligand is bound to a solid phase support, and each column having a inflow end and an outflow end, wherein each of said columns is capable of independently having a target receptor or a plurality of target receptors applied thereto under frontal chromatography conditions to produce an effluent from the outflow end of the column;
b) a plurality of first reservoirs each connected to the inflow end of one of the columns for applying a target receptor or a plurality of target receptors to the columns;
(c) a plurality of second reservoirs each connected to the inflow end of one of the columns for applying either (i) a mixture comprising the target receptor or plurality of target receptors, at least one void marker compound and an indicator compound or a plurality of indicator compounds, (ii) at least one void marker compound and an indicator compound or a plurality of indicator compounds, or (iii) a buffer solution to the column.
(d) a mass spectrometer connected to the outflow end of each of said columns for intermittently analyzing the effluent from each of the column.
In a preferred embodiment, the above apparatus further comprises:
(e) a third reservoir connected to the outflow end of each of the columns for supplying a supplemental diluent to the effluent from each column before analysis by the mass spectrometer.
Preferably, the above described apparatus comprises from 2 to about 100 columns, more preferably from 3 to about 50 columns; and still more preferably from 5 to about 10 columns.
Preferably, each column is intermittently monitored for a period of about 0.5 seconds to about 10 seconds, preferably for about 1 second to about 5 seconds, before switching to the next column.